PROJECT SUMMARY/ABSTRACT ? PROJECT 3 Protective immunity against HIV may be greatly facilitated by the generation of strong immune defenses localized to mucosal portals of entry, where multiple mechanisms of cellular and humoral immunity can participate in virus neutralization. Projects 1 and 2 of this HIVRAD program focus on generating a mechanistic understanding of striking protection observed in macaques through a combination of IgG and IgA antibodies at mucosal surfaces; Project 3 aims to develop immunization methods capable of inducing such immune responses in concert with cellular immunity at mucosal surfaces. We have recently developed a novel strategy to target both polypeptide antigens and molecular adjuvants to lymph nodes, by conjugating these vaccine components to amphiphilic albumin-binding lipid tails, which efficiently causes albumin-mediated lymph node uptake (Liu et al. Nature 2014). This simple modification to create amphiphile (amph)-vaccines increases the T-cell response to molecular vaccines by 30-fold and humoral responses by 20-fold, and greatly increases the therapeutic efficacy of cancer vaccines in mice. This strategy for lymph node targeted adjuvant delivery simultaneously enhances the safety profile of potent molecular adjuvants, by blocking systemic dissemination. It has also recently been demonstrated that mucosal immunization can be dramatically enhanced by exploiting neonatal Fc receptor (FcRn)-mediated transcytosis of Fc-antigen fusion proteins across mucosal barriers. Because albumin is also recycled through FcRn binding, we hypothesize that amph-vaccines will be able to bind to endogenous albumin in interstitial fluid and achieve similar efficient transport across mucosal barriers to permit needle-free mucosal vaccination through the nasal mucosa to promote humoral immunity at distal reproductive tract mucosal sites. Our specific aims are: (1) Synthesize a set of candidate amphiphile-adjuvants for lymph node targeting of mucosa-draining lymph nodes; (2) Determine limits and best strategies for lymph node targeting of antigens; (3) Define pharmacokinetics and early innate immune/toxicity responses of lymph node-targeted vaccines for mucosal vaccination in non-human primates; and (4) Test the capacity of LN- targeted vaccines to enhance the immunogenicity of HIV subunit vaccines, promote mucosal cellular and humoral immunity, and promote protection against mucosal SHIV challenge in non-human primates. Results from this project will establish the translation of ?albumin hitchhiking? as a strategy to potentiate cellular and humoral to HIV at mucosal surfaces in non-human primates, and test the capacity of this promising new approach to maximize protection against heterologous SHIV challenge through induction of polyfunctional immunity at mucosal portals of entry.